Galanin receptor 2 proteins and nucleic acids

ABSTRACT

The present invention is directed to galanin receptor 2 (GAL-R2) proteins and nucleic acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 10/359,285 filed Feb.6, 2003, which is a continuation of U.S. Ser. No. 08/981,700 filed Jan.7, 1998, which issued as U.S. Pat. No. 6,562,945 on May 13, 2003, whichis the U.S. National Stage filing of International Application SerialNo. PCT/SE97/01217 filed Jul. 4, 1997, which claims priority to SwedishApplication SE 9602822-0 filed Jul. 19, 1996.

FIELD OF THE INVENTION

The present invention is in the general field of biological receptorsand the various uses that can be made of such receptors. Morespecifically, the invention relates to nucleic acids encoding a novelgalanin receptor and the receptor protein itself.

BACKGROUND AND PRIOR ART

Galanin is a small (29-30 amino acid) neuroendocrine peptide which doesnot belong to any known peptide family (Bedecs et al., Int. J. Biochem.Cell. Biol. 27: 337-349 (1995)). It is widely distributed in the centralnervous system and other tissues, and has been reported to have a largenumber of diverse biological and pharmacological activities. Galanin hasbeen reported to: (a) promote growth hormone release (Bauer et al., TheLancet 2:192-195 (1986)); (b) inhibit glucose-induced insulin release(Ahren et al., FEBS Lett. 299:233-237 (1988)); (c) regulate motility inthe gastrointestinal tract (Fox-Thelkeld et al., Gastroenter-ology101:1471-1476 (1991)); (d) stimulate feeding behavior (Crawley et al.,J. Neurosci 10:3695-3700 (1990)); and (e) impair cognitive function(Mastropaolo et al., Proc. Nat'l Acad. Sci. USA. 85:9841-9845 (1988)).

Of particular pharmacological interest are galanin's analgesic effects(Post et al., Acta Physiol. Scand. 132:583-584 (1988)). In the spinalcord, galanin inhibits nociceptive reflexes and potentiates theanalgesic effect of morphine (Wiesenfeld-Hallin et al., Neurosci. Lett.105:149-154 (1989)). Target administration of galanin hyperpolarizesdorsal horn neurons and chronic administration of a galanin receptorantagonist after axotomy has been reported to markedly increase autonomyin rats (Verge et al., Neurosci. Lett. 149:193-197 (1993)). Theseobservations indicate that galanin, like morphine, has stronganti-nociceptive actions in vivo. Thus, the known pharmacologicaleffects of galanin suggest potential therapeutic applications as ananesthetic or analgesic in animals and humans.

Galanin exerts its effects by binding to membrane-bound receptors. ThecDNA for one such receptor (“GAL-R1”) has been cloned from both humansand rats (Habert-Ortoliet et al., Proc. Natl. Acad. Sci. USA.91:9780-9783 (1994); Burgevin et al., J. Mol. Neurosci. 6:33-41 (1995)).High levels of rat GAL-R1 mRNA have been found in the ventralhippocampus, thalamus, amygdala, and medulla oblongata of the brain andin the dorsal horn of the spinal cord (Burgevin et al., supra).Pharmacological data obtained using galanin fragments, agonists andantagonists have suggested that more than one type of receptor may beresponsible for galanin's actions (for a review, see Valkna et al.,Neurosci. Lett. 187:75-78 (1995)). The isolation and characterization ofnew receptors for galanin would be highly desirable to assist in thediscovery and development of therapeutic agents for altering galaninactivity in vivo.

SUMMARY OF THE INVENTION

The present invention is based upon the discovery of a novel galaninreceptor (“GAL-R2”) which is distinct from previously reported receptorsin terms of structure, tissue distribution and binding characteristics.Receptors from both the rat and human have been isolated and sequenced.As used herein, the term “GAL-R2” refers to the receptor from either ofthese species unless the text, expressly or by context, indicatesotherwise.

In its first aspect, the invention is directed to proteins, except asexisting in nature, comprising the amino acid sequence consistingfunctionally of rat GAL-R2 (as shown in FIG. 1) or consistingfunctionally of human GAL-R2 (as shown in FIG. 2). The term “consistingfunctionally of” refers to proteins in which the sequence of FIG. 1 orFIG. 2 has undergone additions, deletions or substitutions which do notsubstantially alter the functional characteristics of the receptor.Thus, the invention encompasses proteins having exactly the same aminoacid sequence as shown in the figures, as well as proteins withdifferences that are not substantial as evidenced by their retaining thebasic, qualitative ligand binding properties of GAL-R2. The inventionfurther encompasses substantially pure proteins consisting essentiallyof a GAL-R2 amino acid sequence, antibodies that bind specifically toGAL-R2 (i.e. that have at least a 100 fold greater affinity for GAL-R2than any other protein), and antibodies made by a process involving theinjection of pharmaceutically acceptable preparations of such proteinsinto an animal capable of antibody production. In a preferredembodiment, monoclonal antibody to GAL-R2 is produced by injecting thepharmaceutically acceptable preparation of GAL-R2 into a mouse and thenfusing mouse spleen cells with myeloma cells.

The invention is also directed to a substantially pure polynucleotideencoding a protein comprising the amino acid sequence consistingfunctionally of the sequence of rat GAL-R2 (SEQ ID NO:2 (as shown inFIG. 1) or human GAL-R2 (SEQ ID NO:4) (as shown in FIG. 2). This aspectof the invention encompasses polynucleotides encoding proteinsconsisting essentially of the amino acid sequences in the figures,expression vectors comprising such polynucleotides, and host cellstransformed with such vectors. Also included is the recombinant rat andhuman GAL-R2 proteins produced by host cells made in this manner.Preferably, the polynucleotide encoding rat GAL-R2 has the nucleotidesequence shown in FIG. 1 (SEQ ID NO: 1) and the polynucleotide encodinghuman GAL-R2 has the nucleotide sequence show in-FIG. 2 (SEQ ID NO:3).It is also preferred that the vectors and host cells used for theexpression of GAL-R2 use these particular polynucleotides.

In another aspect, the present invention is directed to a method forassaying a test compound for its ability to bind to GAL-R2. This methodis performed by incubating a source of GAL-R2 with a ligand known tobind to the receptor and with the test compound. The source of GAL-R2should be substantially free of other types of galanin receptors, i.e.greater than 90% of the galanin receptors present should correspond toGAL-R2. Upon completion of incubation, the ability of the test compoundto bind to GAL-R2 is determined by the extent to which ligand bindinghas been displaced. A preferred source of GAL-R2 for use in the assay isa cell transformed with a vector for expressing the receptor andcomprising a polynucleotide encoding a protein consisting essentially ofthe amino acid sequence shown in FIG. 1 (SEQ ID NO:2) (rat GAL-R2) andFIG. 2 (SEQ ID NO:4) (human GAL-R2). Instead of using cells in theassay, a membrane preparation can be prepared from the cells and thiscan be used as the source of GAL-R2. Although not essential, the assaycan be accompanied by the determination of the activation of a secondmessenger pathway such as the adenyl cyclase pathway. This should helpto determine whether a compound that binds to GAL-R2 is acting as anagonist or antagonist to galanin.

In another aspect, the present invention is directed to a method forassaying a test compound for its ability to alter the expression ofGAL-R2. This method is performed by growing cells expressing GAL-R2, butsubstantially free of other galanin receptors, in the presence of thetest compound. Cells are then collected and the expression of GAL-R2 iscompared with expression in control cells grown under essentiallyidentical conditions but in the absence of the test compound. Inpreferred embodiments, the cells expressing GAL-R2 are cells transformedwith an expression vector comprising a polynucleotide sequence encodinga protein consisting essentially of the amino acid sequence shown inFIG. 1 (SEQ ID NO:2) (rat GAL-R2) or FIG. 2 (SEQ ID NO:4) (humanGAL-R2). A preferred test compound is an oligonucleotide at least 15nucleotides in length and comprising a sequence complimentary to asequence shown in one or both of the figures. The preferred method fordetermining receptor expression is by means of a receptor binding assay.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: The composite nucleotide sequence and corresponding translatedamino acid sequence (in single letter code) of rat GAL-R2 is shown. Thenucleic acid sequence has been given the designation SEQ ID NO:1 and theamino acid sequence, SEQ ID NO:2.

FIG. 2: The composite nucleotide sequence and corresponding translatedamino acid sequence (in single letter code) of human GAL-R2 is shown.The nucleic acid sequence has been given the designation SEQ ID NO:3 andthe amino acid sequence, SEQ ID NO:4.

FIG. 3: The amino acid sequences of rat GAL-R2 (RGALR2. PRO) (SEQ IDNO:2), rat GAL-R1 (rGALR1. PRO) (SEQ ID NO:5), human GAL-R2 (HGALR2.PRO) (SEQ ID NO:4) and human GAL-R1 (hGALR1.PRO) (SEQ ID NO:6) arealigned to show regions of homology. The residues in the HGAL-R2sequence that are shared with other sequences are boxed. In order tooptimize alignment, gaps were created at several places in GAL-R2sequences and these gaps are indicated by black boxes.

FIG. 4: The saturation isotherm of ¹²⁵I-galanin binding to membranesfrom rat GAL-R2-expressing HEK-293 cells is shown. Increasingconcentrations of radiotracer were incubated with the membranes, bindingwas allowed to reach equilibrium, and then the reaction was filtered asdescribed under Example 3. Nonspecific binding was measured in thepresence of 1 μM of unlabeled galanin and was subtracted from totalbinding to obtain specific binding.

FIG. 5: FIG. 4 shows the results of binding assays in which unlabeledgalanin and galanin-related peptides were allowed to compete withlabeled galanin for GAL-R2 sites. The data has been converted topercentages, with binding in the absence of competitor serving as 100%.No inhibition was observed when binding assays were performed in thepresence of peptides unrelated to galanin.

FIG. 6: Galanin attenuated the stimulation of adenyl cyclase byforskolin in a dose-dependent manner in HEK-293 cells expressing GAL-R2.Panel A shows the basal level of cAMP in cells not treated with eitherforskolin or galanin (C); the effect of 1 μM galanin (G); the effect of0.1 mM forskolin (F); and the effect of 1 μM galanin+0.1 mM forskolin(F+G). In panel B, cells were incubated in the presence of 0.1 mMforskolin alone or in the presence of forskolin with variousconcentrations of galanin. Intracellular cAMP was then extracted andmeasured by enzyme immunoassay as described in Example 4. Results areexpressed as percentages, where 100% is the value obtained in thepresence of forskolin alone.

DEFINITIONS

The description that follows uses a number of terms that refer torecombinant DNA technology. In order to provide a clear and consistentunderstanding of the specification and claims, including the scope to begiven such terms, the following definitions are provided.

Cloning vector: A plasmid or phage DNA or other DNA sequence which isable to replicate autonomously in a host cell, and which ischaracterized by one or a small number of restriction endonucleaserecognition sites. A foreign DNA fragment may be spliced into the vectorat these sites in order to bring about the replication and cloning ofthe fragment. The vector may contain a marker suitable for use in theidentification of transformed cells. For example, markers may providetetracycline resistance or ampicillin resistance.

Expression vector: A vector similar to a cloning vector but which iscapable of inducing the expression of the DNA that has been cloned intoit, after transformation into a host. The cloned DNA is usually placedunder the control of (i.e., operably linked to) certain regulatorysequences such as promoters or enhancers. Promoter sequences may beconstitutive, inducible or repressible.

Substantially pure: As used herein, “substantially pure” means that thedesired product is essentially free from contaminating cellularcomponents. Contaminants may include, but are not limited to, proteins,carbohydrates or lipids. One method for determining the purity of aprotein or nucleic acid is by electrophoresing a preparation in a matrixsuch as polyacrylamide or agarose. Purity is evidenced by the appearanceof a single band after staining.

Host: Any prokaryotic or eukaryotic cell that is the recipient of areplicable expression vector or cloning vector is the “host” for thatvector. The term encompasses prokaryotic or eukaryotic cells that havebeen engineered to incorporate a desired gene on its chromosome or inits genome. Examples of cells that can serve as hosts are well known inthe art, as are techniques for cellular transformation (see e.g.Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. ColdSpring Harbor (1989)).

Promoter: A DNA sequence typically found in the 5′ region of a gene,located proximal to the start codon. Transcription is initiated at thepromoter. If the promoter is of the inducible type, then the rate oftranscription increases in response to an inducing agent.

Complementary Nucleotide Sequence: A complementary nucleotide sequence,as used herein, refers to the sequence that would arise by normal basepairing. For example, the nucleotide sequence 5′-AGAC-3′ would have thecomplementary sequence 5′-GTCT-3′.

Expression: Expression is the process by which a polypeptide is producedfrom DNA. The process involves the transcription of the gene into mRNAand the translation of this mRNA into a polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the GAL-R2 receptor proteins,genetic sequences coding for the receptors, a method for assayingcompounds for binding to GAL-R2 and a method for assaying compounds fortheir ability to alter GAL-R2 expression. The receptors and theirnucleic acids are defined by their structures (as shown in FIGS. 1 and2) as well as by their tissue distribution and binding characteristics.

With respect to structure, it will be understood that the presentinvention encompasses not only sequences identical to those shown in thefigures, but also sequences that are essentially the same and sequencesthat are otherwise substantially the same and which result in a receptorretaining the basic binding characteristics of GAL-R2. For example, itis well known that techniques such as site-directed mutagenesis may beused to introduce variations in a protein's structure. Variations inGAL-R2 introduced by this or some similar method are encompassed by theinvention provided that the resulting receptor retains the ability tospecifically bind to galanin or galanin-like peptides. Thus, theinvention relates to proteins comprising amino acid sequences consistingfunctionally of the sequence of SEQ ID NO:2 (rat) and SEQ ID NO:4(human).

I. Nucleic Acid Sequences Coding for GAL-R2

DNA sequences coding for GAL-R2 are present in a variety of tissues, anyof which may serve as a source for the isolation of nucleic acid codingfor the receptor. In rats, spinal cord and brain tissues are among thepreferred sources with the dorsal ganglia of the spinal cord and thehippocampus, mammillary bodies and cerebellum of the brain beingespecially preferred. In addition, cells and cell lines that expressGAL-R2 may serve as a source for nucleic acid. These may either becultured cells that have not undergone transformation or cell linesspecifically engineered to express recombinant GAL-R2.

Many methods are available for isolating DNA sequences and may beadapted for the isolation of GAL-R2 nucleic acid (see for exampleSambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Press (1989)). One preferred method for rat GAL-R2,illustrated in Example 1, is to screen a cDNA library that has beenprepared by reverse transcribing mRNA isolated from tissues or cellsknown to express GAL-R2. The library may be prepared from, for example,rat dorsal root ganglia or from brain tissue. A rat brain stem spinalcord cDNA library in ZAP II has been found to produce suitable results.A similar method can be used for human GAL-R2 or, alternatively, a humanDNA library can be screened as described in Example 7.

It is expected that a wide variety of probes specific for GAL-R2 can beused equally well for the screening of cDNA libraries. One way to easilyproduce a large amount of probe is to use the polymerase chain reaction(PCR) to amplify the desired sequence from a cDNA library. For example,PCR may be performed on a cDNA library from rat dorsal root gangliausing the primers:

(SEQ ID NO:7) TM2: 5′-GGCCGTCGACTTCATCGTC(AorT)(AorC)(TorC)CTI(GorT)CI(TorC)TIGC(A, C, GorT)GAC-3′ (SEQ ID NO:8) TM7:5′-(AorG)(C, AorT)(AorT)(AorG)CA(AorG) TAIATIATIGG(AorG)TT-3′

The letter “I” in the sequences above, is the abbreviation for inosine.

Amplified fragments can be size fractionated on an agarose gel and theselected fragments (e.g., fragments 400-1,000 base pairs in length)inserted into an appropriate vector (e.g., pGEM-T). The vector may beintroduced into competent cells (e.g., DH5 cells) by any of theestablished methods for cell transformation, e.g., by calcium phosphateprecipitation. Transformed cells containing the DNA of interest may beidentified by again performing PCR with the TM2 and TM7 primers. The DNAinserts present in these cells are excised, purified and labeled with³²P. The labeled DNA fragments thus produced are used as probes forscreening a cDNA library for GAL-R2. The presence of the correctsequence in selected cells may be confirmed by DNA sequencing and, ifnecessary, partial clones may be spliced together to form a full-lengthsequence.

Although the above procedure is known to be suitable for obtainingGAL-R2 nucleic acid, it is expected that alternative techniques can bedeveloped with relatively little effort. Thus, cDNA libraries may bescreened using probes synthesized based upon the GAL-R2 sequence shownin FIG. 1 for rats and shown in FIG. 2 for humans. In general, probesshould be at least 14 nucleotides long and should not be selected fromregions known to be highly conserved among proteins, e.g., thetransmembrane domains of G-protein linked receptors. Alternatively,using the sequences shown in the figures, it should be possible toselect PCR primers that amplify the full-length GAL-R2 sequence. Thesame techniques that have proven successful in the rat and human can beused to obtain GAL-R2 sequences from other species as well.

II. Production and Isolation of GAL-R2 Recombinant Protein

In order to express recombinant GAL-R2, the structural sequence for theprotein described above must be placed in a vector containingtranscriptional and translational signals recognizable by an appropriatehost. The cloned GAL-R2 sequences, preferably in double-stranded form,are inserted into the expression vector in an operable linkage, i.e.,they are positioned so as to be under the control of the vector'sregulatory sequences and in such a manner that mRNA is produced which istranslated into the GAL-R2 amino acid sequence.

Expression of the GAL-R2 receptor protein in different hosts may resultin different post-translational modifications that can, potentially,alter the properties of the receptor. Preferably, nucleic acid encodingGAL-R2 is expressed in eukaryotic cells, especially mammalian cells.These cells provide post-translational modifications which, inter alia,aid in the correct folding of the receptor protein. Examples of anappropriate vector, pCDNA3-GAL-R2, and host, HEK293 cells, are given inthe Example 2.

Other mammalian cells that may be used include, without limitation,NIH-3T3 cells, CHO cells, HeLa cells, LM(tk-) cells etc. Vectorssuitable for use in each of these various cell types are well known inthe art (see e.g. Sambrook et al., supra). Preferred eukaryoticpromoters include that of the mouse metallothionein I gene; the TKpromoter of Herpes virus; the SV40 early promoter; and the yeast GAL4gene promoter. Some examples of suitable prokaryotic promoters includethose capable of recognizing T4 polymerases, the P_(R) and P_(L)promoters of bacteriophage lambda, and the trp, recA, heat shock andlacZ promoters of E. coli.

Expression vectors may be introduced into host cells by methods such ascalcium phosphate precipitation, microinjection or electroporation.Cells expressing the GAL-R2 receptor can be selected using methods wellknown in the art. One simple method for confirming the presence of thereceptor nucleic acid in cells is to perform PCR amplification using theprocedures and primers discussed above. The presence of functionalreceptor may be confirmed by performing binding assays using labeledgalanin.

Once cells producing recombinant GAL-R2 receptor have been identified,they may be used in either binding assays or in assays designed toidentify agents capable of altering GAL-R2 expression. Alternatively,membranes may be isolated from the cells and used in receptor bindingassays.

III. Antibodies to GAL-R2

The present invention also is directed to antibodies that bindspecifically to GAL-R2 and to a process for producing such antibodies.Antibodies that “bind specifically to GAL-R2” are defined as those thathave at least a one hundred fold greater affinity for GAL-R2 than forGAL-R1 and any undenatured protein not binding galanin. The process forproducing such antibodies may involve either injecting the GAL-R2protein itself into an appropriate animal or, preferably, injectingshort peptides made to correspond to different regions of GAL-R2. Thepeptides should be at least five amino acids in length and should beselected from regions believed to be unique to the GAL-R2 protein. Thus,highly conserved transmembrane regions should generally be avoided inselecting peptides for the generation of antibodies. Methods for makingand detecting antibodies are well known to those of skill in the art asevidenced by standard reference works such as: Harlow et al.,Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.(1988)); Klein, Immunology: The Science of Self-Nonself Discrimination(1982); Kennett, et al., Monoclonal Antibodies and Hybridomas: A NewDimension in Biological Analyses (1980); and Campbell, “MonoclonalAntibody Technology,” in Laboratory Techniques in Biochemistry andMolecular Biology, (1984)).

“Antibody,” as used herein, is meant to include intact molecules as wellas fragments which retain their ability to bind to antigen (e.g., Faband F(ab)₂ fragments). These fragments are typically produced byproteolytically cleaving intact antibodies using enzymes such as papain(to produce Fab fragments) or pepsin (to produce F(ab)₂ fragments). Theterm “antibody” also refers to both monoclonal antibodies and polyclonalantibodies. Polyclonal antibodies are derived from the sera of animalsimmunized with the antigen. Monoclonal antibodies can be prepared usinghybridoma technology (Kohler, et al., Nature 256:495 (1975); Hammerling,et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, M.Y., pp. 563-681 (1981)). In general, this technology involves immunizingan animal, usually a mouse, with either intact GAL-R2 or a fragmentderived from GAL-R2. The splenocytes of the immunized animals areextracted and fused with suitable myeloma cells, e.g., SP₂O cells. Afterfusion, the resulting hybridoma cells are selectively maintained in HATmedium and then cloned by limiting dilution (Wands, et al.,Gastroenterology 80:225-232 (1981)). The cells obtained through suchselection are then assayed to identify clones which secrete antibodiescapable of binding to GAL-R2.

The antibodies, or fragments of antibodies, of the present invention maybe used to detect the presence of GAL-R2 protein using any of a varietyof immunoassays. For example, the antibodies may be used inradioimmunoassays or in immunometric assays, also known as “two-site” or“sandwich” assays (see Chard, T., “An Introduction to Radioimmune Assayand Related Techniques,” in Laboratory Techniques in Biochemistry andMolecular Biology, North Holland Publishing Co., N.Y. (1978)). In atypical immunometric assay, a quantity of unlabelled antibody is boundto a solid support that is insoluble in the fluid being tested, e.g.,blood, lymph, cellular extracts, etc. After the initial binding ofantigen to immobilized antibody, a quantity of detectably labeled secondantibody (which may or may not be the same as the first) is added topermit detection and/or quantitation of bound antigen (see e.g.Radioimmune Assay Method, Kirkham et al., e.d., pp. 199-206, E & S.Livingstone, Edinburgh (1970)). Many variations of these types of assaysare known in the art and may be employed for the detection of GAL-R2.

Antibodies to GAL-R2 may also be used in the purification of either theintact receptor or fragments of the receptor (see generally, Dean etal., Affinity Chromatography, A Practical Approach, IRL Press (1986)).Typically, antibody is immobilized on a chromatographic matrix such asSepharose 4B. The matrix is then packed into a column and thepreparation containing GAL-R2 is passed through under conditions thatpromote binding, e.g., under conditions of low salt. The column is thenwashed and bound GAL-R2 is eluted using a buffer that promotesdissociation from antibody, e.g., buffer having an altered pH or saltconcentration. The eluted GAL-R2 may be transferred into a buffer ofchoice, e.g., by dialysis, and either stored or used directly.

IV. Assay for GAL-R2 Binding

One of the main uses for GAL-R2 nucleic acids and recombinant proteinsis in assays designed to identify agents, other than galanin, capable ofbinding to GAL-R2 receptors. Such agents may either be agonists,mimicking the effects of galanin, or antagonists, inhibiting the effectsof galanin. Of particular interest is the identification of agents whichbind to the GAL-R2 receptors and modulate adenyl cyclase activity in thecells. These agents have potential therapeutic application as eitheranalgesics or anesthetics.

An example of an assay that may be used for detecting compounds bindingto GAL-R2 is presented in Example 4. The essential feature of this assayis that a source of GAL-R2 is incubated together with a ligand known tobind to the receptor and with the compound being tested for bindingactivity. The preferred source for GAL-R2 is cells, preferably mammaliancells, transformed to recombinantly express the receptor. The cellsselected should not express a substantial amount of any other receptorwhich binds galanin, e.g., GAL-R1. This can easily be determined byperforming galanin binding assays on cells derived from the same tissueor cell line as those recombinantly expressing GAL-R2 but which have notundergone transformation.

The assay may be performed either with intact cells or, preferably, withmembranes prepared from the cells (see e.g. Wang, et al., Proc. Natl.Acad. Sci. U.S.A. 90:10230-10234 (1993)). The membranes are incubatedwith a ligand specific for galanin receptors and with a preparation ofthe compound being tested. After binding is complete, receptor isseparated from the solution containing ligand and test compound, e.g. byfiltration, and the amount of binding that has occurred is determined.Preferably, the ligand used is galanin detectably labeled with aradioisotope such as ¹²⁵I. However, if desired, fluorescent orchemiluminescent labels can be used instead. Among the most commonlyused fluorescent labeling compounds are fluorescein isothiocynate,rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehydeand fluorescamine. Useful chemiluminescent compounds include luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt, andoxalate ester. Any of these agents which can be used to detectably labelgalanin will produce a ligand suitable for use in the assay.

Nonspecific binding may be determined by carrying out the bindingreaction in the presence of a large excess of unlabelled ligand. Forexample, ¹²⁵I-galanin may be incubated with receptor and test compoundin the presence of a thousandfold excess of unlabelled galanin.Nonspecific binding should be subtracted from total binding, i.e.binding in the absence of unlabeled galanin, to arrive at the specificbinding for each sample tested. Other steps such as washing, stirring,shaking, filtering and the like may be included in the assays asnecessary. Typically, wash steps are included after the separation ofmembrane-bound ligand from ligand remaining in solution and prior toquantitation of the amount of ligand bound, e.g., by countingradioactive isotope. The specific binding obtained in the presence oftest compound is compared with that obtained in the presence of labeledligand alone to determine the extent to which the test compound hasdisplaced galanin.

In performing binding assays, care must be taken to avoid artifactswhich may make it appear that a test compound is interacting with theGAL-R2 receptor when, in fact, binding is being inhibited by some othermechanism. For example, the compound being tested should be in a bufferwhich does not itself substantially inhibit the binding of galanin toGAL-R2 and should, preferably, be tested at several differentconcentrations. Preparations of test compound should also be examinedfor proteolytic activity and it is desirable that antiproteases beincluded in assays. Finally, it is highly desirable that compoundsidentified as displacing the binding of ligand to GAL-R2 receptor bereexamined in a concentration range sufficient to perform a Scatchardanalysis on the results. This type of analysis is well known in the artand can be used for determining the affinity of a test compounds forreceptor (see e.g., Ausubel, et al., Current Protocols in MolecularBiology, 11.2.1-11.2.19 (1993); Laboratory Techniques and Biochemistryand Molecular Biology, Work, et al., ed., N.Y. (1978) etc.). Computerprograms may be used to help in the analysis of results (see e.g.,Munson, P., Methods Enzymol. 92:543-577 (1983); McPherson, G. A.,Kinetic, EBDA Ligand, Lowry-A Collection of Radioligand Binding AnalysisPrograms, Elsevier-Biosoft, U.K. (1985)). An example of the types ofcurves that may be obtained using this method is shown in FIG. 5 andexamples of inhibitory constants for galanin-related peptidesdeter-mined using binding assays are shown in Table 1.

The activation of a second messenger pathway may be examined byperforming adenyl cyclase assays for compounds that have been identifiedas binding to the GAL-R2 receptor. These assays may be carried out asdiscussed in Example 5 or using any other method for determining cAMPconcentration. Typically, adenyl cyclase assays will be performedseparately from binding assays, but it may also be possible to performbinding and adenyl cyclase assays on a single preparation of cells.

V. Assay for Ability to Modulate GAL-R2 Expression

One way to either increase or decrease the biological effects of galaninis to alter the extent to which GAL-R2 is expressed in cells. Therefore,assays for the identification of compounds that either inhibit orenhance expression are of considerable interest. These assays arecarried out by growing cells expressing GAL-R2 in the presence of a testcompound and then comparing receptor expression in these cells withcells grown under essentially identical conditions but in the absence ofthe test compound. As in the binding assays discussed above, it isdesirable that the cells used be substantially free of receptors forgalanin other than GAL-R2. Scatchard analysis of binding assaysperformed with labeled galanin can be used to determine receptor number.The binding assays may be carried out as discussed above in section IVand will preferably utilize cells that have been engineered torecombinantly express GAL-R2 as described in sections I and II.

A preferred group of test compounds for inclusion in the GAL-R2expression assay consists of oligonucleotides complementary to varioussegments of the GAL-R2 nucleic acid sequence. These oligonucleotidesshould be at least 15 bases in length and should be derived fromnon-conserved regions of the receptor nucleic acid sequence.

Oligonucleotides which are found to reduce receptor expression may bederivatized or conjugated in order to increase their effectiveness. Forexample, nucleoside phosphorothioates may be substituted for theirnatural counterparts (see Cohen, J., Oligodeoxynucleotides, AntisenseInhibitors of Gene Expression, CRC Press (1989)). The oligo-nucleotidesmay be delivered to a patient in vivo for the purpose of inhibitingGAL-R2 expression. When this is done, it is preferred that theoligonucleotide be administered in a form that enhances its uptake bycells. For example, the oligonucleotide may be delivered by means of aliposome or conjugated to a peptide that is ingested by cells (see e.g.,U.S. Pat. Nos. 4,897,355 and 4,394,448; see also non-U.S. patentdocuments WO 8903849 and EP 0263740). Other methods for enhancing theefficiency of oligonucleotide delivery are well known in the art and arealso compatible with the present invention.

Having now described the invention, the same will be more readilyunderstood through reference to the following Examples which areprovided by way of illustration and which are not intended to limit thescope of the invention.

EXAMPLES Example 1 Cloning of Rat Galanin Receptor-2 (GAL-R2)

A PCR-based homology screening strategy was used to isolate novel cDNAsequences encoding G protein-coupled receptors. Sequences likely toencode G protein-coupled receptors were amplified from rat dorsal rootganglia mRNA by reverse transcription PCR using the following primers:

(SEQ ID NO:5) TM2: 5′-GGCCGTCGACTTCATCGTC (A or T)(A or C) (T or C)CTI(G or T)C I (T or C)T I GC(A, C, G or T) GAC-3′ (SEQ ID NO:6) TM7: 5′-(Aor G)(C, A or T)(A or T)(A or G)CA (A or G)TAIATIATIGG(A or G)TT-3′

The templates for PCR amplification were synthesized using a “FirstStrand cDNA Synthesis Kit” (Pharmacia Biotech) and 400 ng of dorsal rootganglia poly A+ RNA. The first strand cDNA thus prepared was diluted twofold with distilled water, heated at 95 C for 3 minutes and quicklychilled on ice. 5 μL of the cDNA thus produced was then amplified with50 pmoles of each of the TM2 and TM7 primers and 2.5 units of Taq DNApolymerase in 50 mM KCl, 1.5 mM MgCl₂, 10 mM Tris (HCl), and 200 μMdNTPs, pH 9.0. The reaction tubes were heated at 95° C. for one min. andthen subjected to 40 cycles of denaturation (95° C./1 min), annealing(45° C./1 min) and extension (72° C./1 min). The final extension wasperformed for 10 min. The amplified fragments were analyzed and sizefractionated on a 1.5% agarose gel. Fragments between 400 bp and 1000 bpin length were excised from the gel, purified using a Sephaglas BandPrepkit from Pharmacia, and inserted into a pGEM-T vector from Promega. Therecombinant plasmids thus produced were used to transform competent DH5cells.

Transformed cells were plated on ampicilline-containing 2YT agar platesand recombinant pGEM-T clones were selected by direct colony PCR usingprimers designed for T7 and SP6 promoters. The PCR conditions wereexactly the same as above except 50 pmole each of T7 and SP6 primerswere used instead of TM2 and TM7 primers. The annealing temperature was50 C and 30 cycles were performed. Plasmid DNA was prepared from theclones containing recombinant plasmids using a “Wizard Miniprep DNAPurification System” (Promega Corporation) starting with 4 ml ofbacterial culture. The DNA sequence from these clones was determinedusing the Sanger dideoxynucleotide chain termination method on denatureddouble-stranded plasmid templates using a T7 sequencing kit fromPharmacia.

The insert DNA fragment of clone 3B-21 was excised from the vector usingSac II and Nde I, isolated on an agarose gel and labeled with ³²P byrandom primed synthesis using a Ready-To-Go DNA labeling kit fromPharmacia. This labeled fragment was used to screen a rat brain stemspinal cord cDNA library in ZAP II (Stratagene). Filters wereprehybridized for 2 hours at 42° C. in 50% formamide, 5×SSC, 5×Denhardt's solution, 1% glycine and 100 μg/ml denatured and shearedsalmon sperm DNA. Hybridization with labeled probe was performed at 42 Cfor 18 hours in a solution containing 50% formamide, 5×SSC, 1×Denhardt's solution, 0.3% SDS and 100 μg/ml denatured and sheared salmonsperm DNA. Filters were rinsed twice in 2×SSC, 0.1% SDS at roomtemperature. They were then washed twice for 15 min in 2×SSC, 0.1% SDSat 42 C, twice for 15 min at 42 C in 0.2×SSC, 0.1% SDS, twice with0.05×SSC, 0.1% SDS at 55 C and finally in the same wash solution at 65C.

Hybridization-positive phages were purified and their inserts rescued byhelper phage mediated excision to yield plasmid DNAs. One clone, 21RSC4,contained the complete coding sequence for the receptor except for 51 bpof the 5′ region. This region was obtained by PCR and was then joined atthe Bsu36I site at nucleotide number 16 of 21RSC4. Thus, 67 bp at the5′-end of the coding region of the clone pBS/GALR-2 arose from aPCR-generated fragment.

Example 2 Structural Characteristics of Rat Galanin Receptor-2

The recombinant plasmid pBS/GALR-2 was found to contain an open readingframe of 372 amino acids, flanked by 3′ and 5′ untranslated regions of,respectively, 289 and 308 bp. The sequence of the open reading frame isshown in FIG. 1 along with the amino acid sequence of the encodedprotein. The protein has a molecular mass of 40,700 daltons. Hydropathyanalysis of the protein is consistent with a topography of seventransmembrane domains, indicative of the G-protein-coupled receptorfamily (Sprengel et al., “Hormone Receptors,” in Handbook of Receptorsand Channels: G Protein-Coupled Receptors, Peroutka, S. J., ed., pp.153-207, CRC Press (1994)). In addition, sequence analysis revealed thatthe open reading frame of pBS/GALR-2 contains several conservedstructural features/residues found among the members of the neuropeptidereceptor family, including: an asparagine in TM1 (Asn43); a leucine(Leu67) and an aspartic acid (Asp 71) in TM2; and an arginine (Arg123)and Tyrosine residue (Tyr124) in TM3. Other features of this GAL-R2receptor gene are: potential sites for N-glycosylation in the aminoterminus (Asn2, Asn11); the presence of several serines and threoninesin the carboxyl terminus; and the presence of a second and thirdintracellular loop, which may serve as potential sites forphosphorylation by protein kinases.

A comparison of the rat GAL-R2 open reading frame with the sequences ofhuman GAL-R2 and GAL-R1 receptors is shown in FIG. 3. Overall, ratGAL-R2 has an identity of about 53% at the nucleotide level and 35.5% atthe amino acid level with rat GAL-R1 (Burgevin et al., J. Mol. Neurosci.6:33-41 (1995)) and 34.8% with human GAL-R1 (Habert-Ortoli et al., Proc.Natl. Acad. Sci. USA 919780-9783 (1994)). However the sequence homologyis higher in the putative transmembrane domains. Respectively, thehomologies between the known rat GAL-R1 and GAL-R2 in TM1 to TM7 are37.5%, 67%, 41.6%, 25%, 50%, 33% and 50%.

Overall, it is apparent that GAL-R2 has a unique sequence that sets itapart from the other G-protein-coupled receptors or other members of theneuropeptide receptor subfamily. The amino acid residues essential forthe binding of galanin to the GAL-R1 receptor have been identified asHis264, His267, Phe282 and, to a lesser extent, Glu271. Only one ofthese residues, corresponding to His264, is conserved in GAL-R2.

Example 3 Recombinant Expression of Rat Galanin Receptor-2

To generate a mammalian expression vector, a 1.4 Kb Hind III-Bst-XIrestriction fragment from pBS/GALR-2 was isolated and subcloned betweenthe Hind III and BstX-I sites of pcDNA3 from In Vitrogen, San Diego,Calif. This expression vector, designated pCDNA3/GALR-2, contains, inaddition to the entire receptor coding sequence, 50 bp of 5′untranslated sequence and 288 bp of 3′ untranslated sequence. PlasmidDNA for further analysis was prepared using the Qiaprep system fromQiagen.

A. Transient Transfection

HEK293s cells were obtained from Cold Spring Harbor laboratory. Theywere maintained in culture medium at 37° C., 5% CO₂ and diluted 10 foldevery 3 days. The cells were inoculated in 80 cm² flasks (2×10⁶ cellsper flask) in Dulbeco's Modified Essential Medium (DMEM, Gibco BRL),supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100μg/ml streptomycin and 0.25 μg/ml fungizone. One day after inoculation,cells were transiently transfected using a modified CaCl₂ method(Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press (1989)) and 30 μg of plasmid DNA per flask. The cellswere harvested 48 hours post transfection for ligand binding or signaltransduction experiments.

B. Stable Transfection

HEK293s cells in 80 cm² flasks were transfected with 30 μgpCDNA3/GALR-2. After 21 days of selection in culture medium containing600 μg/ml G418 resistant colonies were pooled and expanded for theradioligand binding and signal transduction studies.

Example 4 Binding Characteristics of Recombinantly Expressed Rat GAL-R2

A. Methods

A galanin binding assay was performed on the crude membranes preparedfrom pcDNA3/GALR-2 transfected cells. The cells were grown in 150 mmpetri dishes to about 80% confluency. Before harvesting, the cells onthe petri dishes were washed once with cold PBS (Gibco BRL). Cells werethen scraped in ice cold PBS using a Teflon cell scraper. The cells thusharvested were gently centrifuged at 1500×g at 4 C, resuspended inmembrane buffer, “MB” (20 mM HEPES pH 7.5 containing 10 μg/mlbenzamidine, 5 μg/ml leupeptin, 5 μg/ml soybean trypsin inhibitor and0.1 mM phenyl methyl sulfonyl fluoride) and were disrupted with aPolytron at a setting of 20,000 rpm for 30 sec. The disrupted cellsuspension was centrifuged at ˜100,000×g for 60 minutes at 4 C using afixed angle rotor in a Beckman L8-70M Ultracentrifuge. The pellet thusobtained was resuspended in the membrane buffer at a concentration of1.0-1.5 mg/ml, aliquoted and frozen at −80 C until used.

The binding reaction was performed in a total volume of 100 μl ofbinding buffer (MB+0.4% bovine serum albumin) containing 5-10 μgmembrane protein and 0.1 nM ¹²⁵I-galanin (2200 Ci/mmol, Dupont/NEN) withor without unlabeled competitors. Non-specific binding was estimated inthe presence of 1 μM of unlabeled galanin. Binding reactions proceededfor 20 min at room temperature and were stopped by filtration throughUnifilters-96, GF/B filters (Canberra Packard), using the 96-wellFiltermate 196 filtration system from Canberra Packard. Filters werewashed 5 times with 0.5 ml of ice cold 20 mM HEPES pH 7.5. The filterswere dried at 55° C. for one hour and then 100 μl of μScint-20 (CanberraPackard) was added per well. Filters were counted with the Topcountmicroplate counter from Canberra Packard.

B. Results

When transfected into HEK293 cells, pCDNA3/GALR-2 resulted in theexpression of specific ¹²⁵I-galanin binding sites. No specific¹²⁵I-galanin binding sites were generated by the transfection of thevector itself or a control pCDNA3 expression construct encoding adelta-opioid receptor. A pool of stable HEK293 cells expressing theGAL-R2 receptor was generated by selecting pCDNA3/GALR-2 transfectedcells using G418 and binding experiments were performed on the membranesof these cells. An example of the results from a binding experiment isshown in FIG. 4.

A single class of saturable ¹²⁵I-galanin binding site was detected withan estimated Kd for ¹²⁵I-galanin of 1.68±0.43 nM and a Bmax of 1-2pmol/mg of crude protein. Various galanin related peptides were used incompetition experiments performed using ¹²⁵I-galanin as a tracer. Thecompetition curves for these peptides are displayed in FIG. 5 and the Kivalues of the peptides tested are summarized in Table 1.

TABLE 1 The inhibitory constants of galanin-related peptides for ¹²⁵I-galanin binding at GAL-R2 PEPTIDE Ki [M] Galanin 2.65 ± 0.07 · 10⁻⁹Galanin(1-16) 1.23 ± 0.70 · 10⁻⁸ M15 3.68 ± 1.20 · 10⁻⁸ M40 8.30 ± 0.49· 10⁻⁹ C7 1.89 ± 1.34 · 10⁻⁷

The binding of labeled galanin was displaced by galanin and galaninrelated peptides but not by galanin unrelated ligands (e.g. substance P,vasoactive intestinal polypeptide, angiotensin II and dynorphin). Themain difference between rat GAL-R1 and GAL-R2, however, lies in therecognition of the chimeric peptide C7, which is equipotent to galaninat the GAL-R1 receptor but is much less active at GAL-R2.

Example 5 Activation of cAMP

Stable pools of transfected cells were inoculated in 24 well plates andallowed to grow overnight. Before experiments, the cells were washedwith PBS at 37 C and then covered with PBS containing 1 mM3-isobutyl-1-methylxanthine (IBMX). Cells in duplicate wells werestimulated for 10 minutes at 37 C either with forskolin (0.1 mM) alone,or in the presence of various concentrations of galanin orgalanin-related peptides. cAMP was extracted in ethanol, lyophilized andresuspended in 0.5 mM assay buffer. Assay of cAMP was performed usingeither the Biotrack cAMP Enzyme-immunoassay System (Amersham) or theCyclic AMP [³H] Assay System (Amersham).

It was found that the activation of rat GAL-R2 in stably transfectedHEK293 cells leads to a significant inhibition of forskolin-stimulatedaccumulation of cAMP and that this inhibition occurs in aconcentration-dependent manner (FIG. 6). Untransfected cells failed toexhibit this effect.

Example 6 In Situ Hybridization

A. Methods

Adult male Sprague-Dawley rats (˜300 gm; Charles River, St-Constant,Quebec) were sacrificed by decapitation. Brain, pituitary and spinalcord were promptly removed, snap-frozen in isopentane at −40 C for 20 sand stored at −80 C. Frozen tissue was sectioned at 14 μm in a Microm HM500 M cryostat (Germany) and thaw-mounted onto ProbeOn Plus slides(Fisher Scientific, Montreal, Quebec). Sections were stored at −80 Cprior to in situ hybridization.

The plasmid pCDNA3-GALR-2 was linearized using either XbaI or HindIIIrestriction enzymes which cut in the polylinker on either side of theinserted cDNA. Sense and antisense GAL-R2 riboprobes were transcribed invitro using either T7 or SP6 RNA polymerases (Pharmacia Biotech), in thepresence of [³⁵S]UTP (˜800 Ci/mmol; Amersham, Oakville, Ontario).Following transcription, the DNA template was digested with DNAse I(Pharmacia). Riboprobes were subsequently purified byphenol/chloro-form/isoamyl alcohol extraction and precipitated in 70%ethanol containing ammonium acetate and tRNA. The quality of labeledriboprobes was verified by polyacrylamide-urea gel electrophoresis.

Sections were postfixed in 4% paraformaldehyde (BDH, Poole, England) in0.1 M phosphate buffer (pH 7.4) for 10 min at room temperature (RT) andrinsed in 3 changes of 2× standard sodium citrate buffer (SSC: 0.15 MNaCl. 0.015 M sodium citrate, pH 7.0). Sections were then equilibratedin 0.1 M triethanolamine, treated with 0.25% acetic anhydride intriethanolamine, rinsed in 2×SSC and dehydrated in an ethanol series(50-100%). Hybridization was performed in a buffer containing 75%formamide, 600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 1× Denhardt'ssolution, 50 mg/ml denatured salmon sperm DNA, 50 mg/ml yeast tRNA, 10%dextran sulfate, 20 mM dithiothreitol and [³⁵S]UTP-labeled cRNA probes(10×10⁶ cpm/ml) at 55 C for 18 h in humidified chambers. Followinghybridization, slides were rinsed in 2×SSC at RT, treated with 20 mg/mlRNase IA in RNase buffer (10 mM Tris, 500 mM NaCl, 1 mM EDTA, pH 7.5)for 45 min at RT and washed to a final stringency of 0.1×SSC at 65 C.Sections were then dehydrated and exposed to Kodak Biomax MR film for 10days and/or dipped in Kodak NTB2 emulsion diluted 1:1 with distilledwater and exposed for 3-4 weeks at 4 C prior to development andcounterstaining with cresyl violet acetate. Neuroanatomical structureswere identified according to the Paxinos and Watson rat brain atlas(Paxinos et al., The Rat Brain in Stereotaxic Coordinates, AcademicPress, N.Y. (1986)).

B. Results

The highest levels of rat GAL-R2 mRNA expression were observed in dorsalroot ganglia with large, intermediate and small diameter cells beingspecifically labeled. Only diffuse labeling was observed throughout thedorsal and ventral horns of the spinal cord. In the rat brain, thehighest densities of GAL-R2 mRNA labeling were detected in the dorsalhippocampus, mammillary bodies and cerebellum (in particular, Purkinjecell layer). More moderate labeling was detected in the pontine nucleusas well as in a specific cranial motor nucleus. Moderate to weakhybridization was detected throughout the cerebral cortices. Othercephalic areas such as the thalamus, the remaining hypothalamus, andbasal ganglia were generally devoid of labeling. This distributiondiffers considerably from that reported for GALR-1 mRNA which isparticularly well expressed in the ventral hippocampus, amygdala,supraoptic nucleus, several hypothalamic and thalamic nuclei, lateralparabrachial nucleus and locus coeruleus of rat brain.

The high level of GAL-R2 expression observed in dorsal root gangliasensory neurons and more moderate levels observed in dorsal horn of thespinal cord is consistent with galanin's role in pain transmission. Thepresence of high levels of GAL-R2 in dorsal hippocampus and mammillarybodies is consistent with a role in cognitive function.

Example 7 Cloning and Structural Features of Human Galanin Receptor-2

A human genomic DNA library prepared from human placenta (Clonetech) inEMBL-3 vector was screened with a random labeled fragment (labeled withT7-Quick-Prime labeling kit cat. #27-9252-01, Pharmacia Biotech.)containing the complete coding region of rat GALR-2 cDNA. Theprehybridization and hybridization conditions were as follows:

Prehybridization: 50% formamide, 5× Denhardt's solution, 5×SSC, 1%glycine, 100 g/ml sheared and denatured salmon sperm DNA at 42° C. for 5hours.

Hybridization: 50% formamide, 1× Denhardt's solution, 5×SSC, 0.3% SDS,100 g/ml sheared and denatured salmon sperm DNA overnight at 42° C.

Wash: A wash step was performed from the low stringency of 2×SSC, 0.1%SDS at 42° C. to the highest stringency of 0.2×SSC, 0.1% SDS at 60° C.(65° C. for Southern blots).

Eight positive clones were identified which were processed for secondaryscreening under hybridization and washing conditions identical to thefirst. The secondary screening resulted in identification of fourclones; the other four clones were considered false-positive. The fourpositive clones were processed for tertiary and quarternary screening inorder to obtain pure clones.

DNA was purified from the four pure clones discussed above and wasprocessed for restriction analysis and Southern blot hybridization inorder to identify smaller fragments which yield a positive signal. Threepositively hybridizing bands (of estimated sizes ˜5 kb, ˜3.2 kb and ˜0.7kb) generated by the cleavage with Sac I and Rsa I restrictionendonucleases (Pharmacia Biotech.) were identified by Southern blothybridization. These bands were excised from the gel and subcloned intoeither Sac I or Eco RV digested pBlueScript KS(-) plasmid. The plasmidconstructs were subjected to sequencing by Sanger dideoxy sequencingmethod (T7 Sequencing kit, Pharmacia Biotech. Cat. #27-1682-01) and theABI Prizm Cycle Sequencing Kit (Cat. #402079, Perkin-Elmer) and thecomposite sequence was constructed.

The nucleotide sequence for human GALR-2 gene is depicted in FIG. 2. Anopen reading frame of 1155 nucleotides is present putatively encoding aprotein of 385 amino acids with a calculated molecular mass of 41478 kD.There is a putative intron of more than 1000 nucleotide in length afterbase number 420. The intronic sequence has been removed from thefinalized sequence reproduced in FIG. 2. The exon-intron boundaries weredetermined based upon the consensus sequences around 5′ and 3′ splicesites in vertebrate pre-mRNAs (Lodish et al. Molecular Cell Biology3^(rd) Ed. Scientific American Books, pp 500; FIG. 4). At the proteinlevel, 84.4% amino acids are identical between rat and human GALR-2; theidentity between the human GALR-2 and the rat or human GALR-1 is about34%.

All references cited herein are fully incorporated by reference. Havingnow fully described the invention, it will be understood by one of skillin the art that the invention may be performed within a wide andequivalent range of conditions, parameters and the like, withoutaffecting the spirit or scope of the invention or any embodimentthereof.

Deposit of Biological Material

The plasmid HUMAN GALR-2 has been deposited under the Budapest Treaty at“Deutsche Sammlung von Mikroorganismen und Zellkulturen” (DSMZ),Braunschweig, Germany. The deposit number is DSM 11632, and the date ofdeposit is 26 Jun. 1997.

1. A substantially pure polynucleotide encoding a protein comprising SEQID NO:2.
 2. A polynucleotide of claim 1 wherein the polynucleotidecomprises SEQ ID NO:1.
 3. A vector comprising the polynucleotide ofclaim
 1. 4. A vector comprising the polynucleotide of claim
 2. 5. A hostcell comprising the vector of claim
 3. 6. A host cell comprising thevector of claim
 4. 7. A substantially pure polynucleotide encoding aprotein comprising SEQ ID NO:4.
 8. A polynucleotide of claim 7 whereinthe polynucleotide comprises SEQ ID NO:3.
 9. A vector comprising thepolynucleotide of claim
 7. 10. A vector comprising the polynucleotide ofclaim
 8. 11. A host cell comprising the vector of claim
 9. 12. A hostcell comprising the vector of claim 10.